In sample analysis instrumentation, and especially in separation systems such as liquid chromatography and capillary electrophoresis systems, smaller dimensions generally result in improved performance characteristics and at the same time result in reduced analysis costs. These techniques are preferably performed using capillaries having small inside diameters ranging from 5 to 100 micrometers for handling the extremely small volumes of flowing liquid samples.
As an example, the micro-column liquid chromatography (.mu.LC) has been described wherein columns having diameters of 100-200 .mu.m are employed as compared to columns having diameters of around 4.6 mm.
Another approach has been the use to capillary electrophoresis, a separation technique carried out in capillaries having a diameter of 5-100 .mu.m.
The capillary electrophoresis has been demonstrated to be useful as a method for the separation of small solutes. See for example Journal of Chromatography, vol. 218, p. 209, 1981; and Analytical Chemistry, vol. 53, p. 1298, 1981.
However, there remain several major problems inherent to those technologies.
For instance, there exist substantial detection limitations in conventional capillary electrophoresis technology. For example in the case of the capillary electrophoresis, optical detection is generally performed on-column by a single-pass detection technique wherein electromagnetic energy, in the form of a light beam is passed through the sample, the light beam travels normal to the capillary axis and crosses the capillary only once. Accordingly, in conventional capillary electrophoresis systems, the detection path length is inherently limited by the diameter of the capillary.
According to Beer's law, absorbance is related to pathlength by the following equation: EQU A=e*b*C
where:
A=the absorbance PA1 e=the molar absorptivity, (l/m*cm) PA1 b=pathlength (cm) PA1 c=concentration (m/l) PA1 .epsilon.(.lambda.)=extinction of molar absorption coefficient as a function of the wavelength, PA1 C=molar solute concentration, PA1 D=pathlength, PA1 I.sub.O =incident photon flux, PA1 I=transmitted photon flux.
From the above equation it is readily understood that the absorbance (A) of a sample in a 25 .mu.m capillary is 400 times lower than to the absorbance of a conventional cell having a pathlength of 1 cm, as typically used in ultraviolet, visible (UV/Vis) spectroscopy.
Standard detection cells used in conventional liquid chromatography instruments have the disadvantage that their cell volume is about of three orders of magnitude too large to handle the small nano-liter (nl) sample volumes of the above mentioned capillary separation techniques. Scaling down the conventional shape of these cells to match the nl volumes is problematic both from the manufacturing point of view and from the photometric aspect. For example, to get a cell volume of approximately 10 nl with a pathlength of 1 mm, the circular inner diameter of such a cell has to be smaller than 120 .mu.m.
With standard UV and UV/Vis detection approaches using conventional lamps such as deuterium lamps or Xenon flash-lamps, it is difficult to pass enough light through such a narrow channel. The loss in light throughput counteracts any gain in optical pathlength, and a sensitivity enhancement over on column detection is no longer possible.
Furthermore, with the state of the art machining tools, e.g., drilling, milling or ultrasonic machining tools it is very difficult to scale down the size of these detection cells to meet these requirements of providing cell volumes in the preferred range of 3-15 nl. Thus, most of the presently available instruments use the above mentioned on column detection approach.
In light of this significant detection limitation, there have been a number of attempts employed in the prior art to extend detection pathlengths, and hence the sensitivity of the analysis in capillary electrophoresis systems.
U.S. Pat. No. 5,061,361 describes an approach entailing micro-manipulation of the capillary flow cell to form a bubble at the point of detection.
U.S. Pat. No. 5,141,548 describes the use of a capillary having a Z-shaped configuration with detection performed across the extended portion of the Z.
Yet another approach has sought to increase the detection pathlength by detecting along the major axis of the capillary (axial-beam detection). See Xi et al., Analytical Chemistry, vol. 62, p. 1580, 1990.
U.S. Pat. No. 5,273,633 describes a further approach to increased detection pathlengths in the field of capillary electrophoresis where a reflecting surface exterior of the capillary is provided. The system also includes an incident window and an exit window downstream of the incident window. Light entering the incident window passes through a section of the capillary by multiple internal reflections before passing through the exit window where it is detected. The multiple internal reflections yield an effective increase in pathlength.
In all of these approaches parts of the light beam do not propagate through the center of the capillary. In all of these approaches at least part of the beam propagates through the transparent capillary wall, and does not contact with the sample. This decreases the linearity of the detector and reduces the effective path length, which determines the sensitivity of the detector.
The European patent application No. 92112114.1 describes a photometric apparatus, having a flow cell coated with a fluoropolymer to guide the light beam axially along a liquid filled tube or capillary. In order to get total internal reflection of the light beam at the cell wall, the fluoropolymer must have a refractive index lower the refractive index of the liquid in the tube. Although these polymers are available, they are very expensive and the stability of the coating when exposed to solvents and acids limits its usability.
While each of the aforementioned approaches has addressed the issue of extending the pathlength, each approach is limited because it entails engineering the capillary after-the-fact or otherwise use difficult technical approaches.
On column detection approaches often use setups similar to those shown in FIG. 8 and FIG. 9.
As shown in FIG. 8 and FIG. 9, in a small area near the outlet a capillary is illuminated with light from a UV/vis light source and a slit of width o is placed behind the capillary to block stray light through the capillary wall. The light transmitted through the slit is detected. Using the Lambert-Beer law, the detector signal for an ideal cell is determined by: ##EQU1## where: A(.lambda.)=absorbance in absorbance units (AU) as a function of the wavelength,
If stray light through the column wall reaches the detector, the above equation is rewritten as: ##EQU2## where: I.sub.S =stray light through the capillary wall.
FIG. 10 is a typical plot of detector response, plotted as absorbance (A(.lambda.)) vs. molar solute concentration (C). The lower detection limit is determined by the baseline noise of the detector and its sensitivity, as determined by the slope dA/dc of the detector response linear range absorbance values in the FIG. 10 plot.
The upper limit of detection is determined by the where the detector response is no longer linear, as occurs at higher concentration levels.
Unwanted stray light causes a deviation from the theoretical linear slope according to the Lambert-Beer law. The steepness of the slope compared to typical HPLC detection cells (HPLC=High Pressure Liquid Chromatography) depends on the effective path length (related to the inside diameter of the capillary) and the stray light through the capillary wall. As a result, there is a linear range in which the detector can be operated to get reliable results.
FIG. 11 is a cross sectional view of a prior art flow cell used in liquid chromatography.
The cell assembly 1100 is positioned between a radiation source 1102 and a photodetector device 1104. A lens 1106 is arranged between the radiation light source 1102 and the cell assembly 1100 for focusing the radiation emitted from the radiation source 1102.
A cell body 1108 include a machined conduit 1110 for holding a liquid sample. Windows or lenses 1112 and 1114 are sealed to the cell body 1108 by gaskets 1116. Screws 1118 and 1120 press the windows 1112, 1114 against the cell body 1108.
An inlet tube 1122 and an outlet tube 1124 are connected by threaded fittings 1126 to an inlet port 1128 and an outlet port 1130, respectively, to supply the sample to and from the cell 1100. The tubes 1122, 1124 normally made of stainless steel are connected by threaded fittings 1126 to the cell body 1108.
Typically, these cells 1100 have cell volumes 1110 in the range of 3 to 15 .mu.l. The cell 1100 has an inner diameter of 0.5 to 1.5 mm and a length of 3 to 10 mm. Because the inner diameter of the cell 1100 is such that light rays are prevented from striking the cell wall, there are unpredictable disturbances of the measured photometric signal. Apertures, conical cell shapes and/or lenses are also used to avoid this problem.
Most commonly, deuterium lamps are used as radiation source 1102 for UV/Vis absorbance measurements.
In capillary electrophoresis and .mu.-liquid chromatography, the sample volumes are about three orders of magnitude smaller than described above. Accordingly, such a flow cell must be much smaller.
Scaling down prior art flow cells of the type described above does not work, since machining operations, e.g. drilling or ultrasonic machining, are not suitable for such small dimensions.
Furthermore, the light throughput through such a small cell is dramatically reduced if no additional changes are made.
Reduced light throughput results in increased photometric noise, to prevent the desired gain in sensitivity from being achieved.